Dengue Fever: Investigation Protocol

We continue with our Back to College Series on Dengue Fever by talking about the Investigation Protocol for Dengue Fever.

The Protocol Consists of Two Parts:

  1. Diseases are monitoring test.
  2. Diagnostic test

Diseases are Monitoring Test

WBC/HCT/Platelets counts/LFT/serum proteins/RFT.

USG abdomen/AXR/ C- reactive proteins/ urine output.

  • Leukopenia followed with progressive thrombocytopenia is suggestive of Dengue infection.
  • Raising HCT accompanying progressive thrombocytopenia is an early sign of DHF.

Recommendations:

  • The complete baselines haemogram should be established as earliest as possible in all suspected patients of dengue. (Grade A)
  • Serial WBC/HCT/platelet to be monitored as diseases progress.(Grade A). A hematocrit level increase greater than 20% is a sign of hemoconcentration and precedes shock.
  • Thrombocytopenia has been demonstrated in up to 50% of dengue fever cases. Platelet counts less than 100,000 cells/μL are seen in dengue hemorrhagic fever or dengue shock syndrome and occur before defervescence and the onset of shock.
  • The platelet count should be monitored at least every 24 hours to facilitate early recognition of dengue hemorrhagic fever and every 3-4 hours in severe cases of dengue hemorrhagic fever or dengue shock syndrome.

Metabolic Panel and Liver Enzymes

  1. Transaminase levels may be mildly elevated into the several thousand in patients with dengue hemorrhagic fever who have acute hepatitis. Low albumin levels are a sign of hemoconcentration.
  2. Elevated blood urea nitrogen (BUN) levels are observed in those with shock. Acute kidney injury is uncommon.
  3. Metabolic acidosis is observed in those with shock and must be corrected rapidly.
  4. Hyponatremia is the most common electrolyte abnormality in patients with dengue hemorrhagic fever or dengue shock syndrome.

Coagulation Studies

Coagulation studies may help to guide therapy in patients with severe hemorrhagic manifestations. Findings are as follows:

  • Prothrombin time is prolonged
  • Activated partial thromboplastin time is prolonged
  • Low fibrinogen and elevated fibrin degradation product levels are signs of disseminated intravascular coagulation

CXR, AXR, USG

CXR, AXR and USG are necessary to pick up fluids accumulation.

Ultrasonography

Ultrasonography is a potentially timely, cost-effective, and easily used modality in the evaluation of potential dengue hemorrhagic fever. Positive and reliable ultrasonographic findings include fluid in the chest and abdominal cavities, pericardial effusion, and a thickened gallbladder wall. Thickening of the gallbladder wall may presage clinically significant vascular permeability.

The utility of previous studies was limited because patients underwent only a single scan. However, in a study by Srikiatkhachorn et al., daily serial ultrasonographic examinations of the thorax and abdomen proved useful in the evaluation of patients with suspected dengue hemorrhagic fever.

Plasma leakage was detected in some patients within three days of fever onset. Pleural effusion was the most common sign. Based on ultrasonographic findings, dengue hemorrhagic fever was predicted in 12 patients before hemoconcentration criteria had been met.

Diagnostic Tools for Dengue Fever

Dengue can be diagnosed by isolation of the virus, by serological tests, or by molecular methods. Diagnosis of acute (on-going) or recent dengue infection can be established by testing serum samples during the first five days of symptoms and early convalescent phase (more than five days of symptoms).

Acute infection with dengue virus is confirmed when the virus is isolated from serum or autopsy tissue specimens, or the specific dengue virus genome is identified by reverse transcription-polymerase chain reaction (RT–PCR) from serum or plasma, cerebrospinal fluid, or autopsy tissue specimens during an acute febrile illness.

Methods such as one-step, real-time RT–PCR or nested RT–PCR are now widely used to detect dengue viral genes in acute-phase serum samples. This detection coincides with the viremia and the febrile phase of illness onset. Acute infections can also be laboratory confirmed by identification of dengue viral antigen or RNA in autopsy tissue specimens by immunofluorescence or immunohistochemical analysis, or by seroconversion from negative to positive IgM antibody to dengue or demonstration of a fourfold or greater increase in IgG antibody titers in paired (acute and convalescent) serum specimens.

Dengue Serology Tests

  • Haemagglutination Inhibition Test
  • Dengue IgM Test
  • Indirect IgG ELISA Test
  • Dengue Rapid Tests
  • NON STRUCTURAL PROTEIN – 1 (NS1 Antigen)
  • POLYMERASE CHAIN REACTION (PCR)
  • VIRUS ISOLATION

Hemagglutination Inhibition Testes

  • Gold Standard for serological diagnosis
  • Labour intensive & requires paired samples for interpretation
  • Differentiate between 1° & 2 ° dengue infection
  • Used mainly for research purpose

Don’t discriminate between other flaviviruses.

Known positive and negative sample should be used.

The haemagglutination-inhibition (HI) test is based on the ability of dengue antigens to agglutinate red blood cells (RBC) of ganders or trypsinized human O RBC. Anti-dengue antibodies in sera can inhibit this agglutination, and the potency of this inhibition is measured in an HI test.

Serum samples are treated with acetone or kaolin to remove non-specific inhibitors of haemagglutination and then adsorbed with gander or trypsinized type O human RBC to remove non-specific agglutinins. Each batch of antigens and RBC is optimized. PH optima of each dengue haemagglutinin require the use of multiple different pH buffers for each serotype.

Optimally the HI test requires paired sera obtained upon hospital admission (acute) and discharge (convalescent) or paired sera with an interval of more than seven days. The assay does not discriminate between infections by closely related fl aviviruses (e.g., between dengue virus and Japanese encephalitis virus or West Nile virus) nor between immunoglobulin isotypes.

The response to a primary infection is characterized by the low level of antibodies in the acute-phase serum drawn before day 5 and a slow elevation of HI antibody titers after that. During secondary dengue infections, HI antibody titers rise rapidly, usually exceeding 1:1280. Values below this are observed in convalescent sera from patients with primary responses.

MAC-ELISA

For the IgM antibody-capture, enzyme-linked immunosorbent assay (MAC-ELISA) total IgM in patients’ sera is captured by anti-µ chain specific antibodies (specific to human IgM) coated onto a microplate. Dengue-specific antigens, from one to four serotypes (DEN-1, -2, -3, and -4), are bound to the captured anti-dengue IgM antibodies and are detected by monoclonal or polyclonal dengue antibodies directly or indirectly conjugated with an enzyme that will transform a non-colored substrate into colored products.

The optical density is measured by a spectrophotometer. Serum, blood on filter paper and saliva, but not urine, can be used for detection of IgM if samples are taken within the appropriate time frame (five days or more after the onset of fever). Serum specimens may be tested at a single dilution or multiple dilutions. Most of the antigens used for this assay are derived from the dengue virus envelope protein (usually virus-infected cell culture supernatants or suckling mouse brain preparations).

MAC-ELISA has good sensitivity and specificity but only when used five or more days after the onset of fever. Different commercial kits (ELISA or rapid tests) are available but have variable sensitivity and specificity. A WHO/TDR/PDVI laboratory network recently evaluated selected commercial ELISAs and first-generation rapid diagnostic tests, finding that ELISAs performed better than rapid tests. Cross-reactivity with other circulating flaviviruses such as Japanese encephalitis, St Louis encephalitis and yellow fever, does not seem to be a problem but some false positives were obtained in sera from patients with malaria, leptospirosis and past dengue infection.

Limitations

These limitations have to be taken into account when using the tests in regions where these pathogens co-circulate. It is recommended that tests be evaluated against a panel of sera from relevant diseases in a particular region before being released to the market. It is not possible to use IgM assays to identify dengue serotypes as these antibodies are broadly cross-reactive even following primary infections. Recently, some authors have described MAC-ELISA that could allow serotype determination, but further evaluations are required.

IgG ELISA

The IgG ELISA used for the detection of a past dengue infection utilizes the same viral antigens as the MAC ELISA. This assay correlates with the hemagglutination assay (HI) previously used. In general IgG ELISA lacks specificity within the flavivirus serocomplex groups.

Primary versus secondary dengue infection can be determined using a simple algorithm. Samples with a negative IgG in the acute phase and a positive IgG in the convalescent phase of the infection are primary dengue infections.

Samples with a positive IgG in the acute phase and a fourfold rise in IgG titter in the convalescent phase (with at least a seven day interval between the two samples) is a secondary dengue infection.

Dengue Rapid Tests

  • Qualitative detection of dengue IgM & IgG
  • The possibility of False Positive results.
  • Need to confirm by dengue IgM ELISA tests

Graph of when laboratory tests for dengue fever become positive. Day zero refers to the start of symptoms, 1st refers to in those with a primary infection, and 2nd refers to in those with a secondary infection.

Detection of Antigens

Until recently, detection of dengue antigens in acute-phase serum was rare in patients with secondary infections because such patients had pre-existing virus-IgG antibody immunocomplexes.

New developments in ELISA and dot blot assays directed to the envelop/membrane (E/M) antigen and the non-structural protein 1 (NS1) demonstrated that high concentrations of these antigens in the form of immune complexes could be detected in patients with both primary and secondary dengue infections up to nine days after the onset of illness.

The NS1 glycoprotein is produced by all flaviviruses and is secreted from mammalian cells. NS1 produces a very strong humoral response. Many studies have been directed at using the detection of NS1 to make an early diagnosis of dengue virus infection. Commercial kits for the detection of NS1 antigen are now available, though they do not differentiate between dengue serotypes.

Performance and Utility

Their performance and utility are currently being evaluated by laboratories worldwide, including the WHO/TDR/PDVI laboratory network. Fluorescent antibody, immune peroxidase and avidin-biotin enzyme assays allow detection of dengue virus antigen in acetone-fixed leucocytes and in snap-frozen or formalin-fixed tissues collected at autopsy.

Dengue RT-PCR

Since the 1990s, several reverse transcriptase-polymerase chain reactions (RT-PCR) assays have been developed. They offer better sensitivity compared to virus isolation with a much more rapid turnaround time. In situ RT-PCR offers the ability to detect dengue RNA in paraffin-embedded tissues. A combination of the four serotype-specific oligonucleotide primers in a single reaction tube (one-step multiplex RT-PCR) is an interesting alternative to the nested RT-PCR.

The products of these reactions are separated by electrophoresis on an agarose gel, and the amplification products are visualized as bands of different molecular weights in the agarose gel using ethidium bromide dye, and compared with standard molecular weight markers. In this assay design, dengue serotypes are identified by the size of their bands.

Compared to virus isolation, the sensitivity of the RT-PCR methods varies from 80% to 100% and depends on the region of the genome targeted by the primers, the approach used to amplify or detect the PCR products (e.g., one-step RT-PCR versus two-step RT-PCR), and the method employed for subtyping (e.g. nested PCR, blot hybridization with specific DNA probes, restriction site-specific PCR, sequence analysis, etc.).

To avoid false positive results due to non-specific amplification, it is important to target regions of the genome that are specific to dengue and not conserved among flavor- or other related viruses.

False-positive results may also occur as a result of contamination by amplicons from previous amplifications. This can be prevented by physical separation of different steps of the procedure and by adhering to stringent protocols for decontamination.

Real-time RT-PCR

The real-time RT-PCR assay is a one-step assay system used to quantitate viral RNA and using primer pairs and probes that are specific to each dengue serotype. The use of a fluorescent probe enables the detection of the reaction products in real time, in a specialized PCR machine, without the need for electrophoresis. Many real-time RT-PCR assays have been developed employing TaqMan or SYBR Green technologies.

The TaqMan real-time PCR is highly specific due to the sequence-specific hybridization of the probe. Nevertheless, primers and probes reported in publications may not be able to detect all dengue virus strains: the sensitivity of the primers and probes depends on their homology with the targeted gene sequence of the particular virus analyzed.

The SYBR green real-time RT-PCR has the advantage of simplicity in primer design and uses universal RT-PCR protocols but is theoretically less specific. Real-time RT-PCR assays are either “singleplex” (i.e., detecting only one serotype at a time) or “multiplex” (i.e., able to identify all four serotypes from a single sample).

The multiplex assays have the advantage that a single reaction can determine all four serotypes without the potential for introduction of contamination during manipulation of the sample.

However, the multiplex real-time RT-PCR assays, although faster, are currently less sensitive than nested RT-PCR assays. An advantage of this method is the ability to determine viral titer in a clinical sample, which may be used to study the pathogenesis of dengue disease.

Isothermal amplification methods

The NASBA (nucleic acid sequence based amplification) assay is an isothermal RNA-specific amplification assay that does not require thermal cycling instrumentation. The initial stage is a reverse transcription in which the single-stranded RNA target is copied into a double-stranded DNA molecule that serves as a template for RNA transcription.

Detection of the amplified RNA is accomplished either by electro chemiluminescence or in real-time with fluorescent-labeled molecular beacon probes. NASBA has been adapted to dengue virus detection with sensitivity near that of virus isolation in cell cultures and may be a useful method for studying dengue infections in field studies. Loop-mediated amplification methods have also been described but their performance compared to other nucleic acid amplification methods are not known.

Virus Isolation

Specimens for virus isolation should be collected early in the course of the infection, during the period of viremia (usually before day 5). The virus may be recovered from serum, plasma and peripheral blood mononuclear cells and attempts may be made from tissues collected at autopsy (e.g., liver, lung, lymph nodes, thymus, bone marrow).

Because dengue virus is heat-labile, specimens awaiting transport to the laboratory should be kept in a refrigerator or packed in wet ice. For storage up to 24 hours, specimens should be kept at between +4 °C and +8 °C. For longer storage, specimens should be frozen at -70 °C in a deep-freezer or stored in a liquid nitrogen container. Storage even for short periods at –20 °C is not recommended.

Importance of Cell Culture

Cell culture is the most widely used method for dengue virus isolation. The mosquito cell line C6/36 (cloned from Ae. albopictus) or AP61 (cell line from Ae. pseudoscutellaris) are the host cells of choice for routine isolation of dengue virus.

Since not all wild-type dengue viruses induce a cytopathic effect in mosquito cell lines, cell cultures must be screened for specific evidence of infection by an antigen detection immunofluorescence assay using serotype-specific monoclonal antibodies and flavivirus group-reactive or dengue complex-reactive monoclonal antibodies. Several mammalian cell cultures, such as Vero, LLCMK2, and BHK21, may also be used but are less efficient.

Virus isolation followed by an immunofluorescence assay for confirmation requires 1–2 weeks and is possible only if the specimen is properly transported and stored to preserve the viability of the virus in it.

When no other methods are available, clinical specimens may also be inoculated by intracranial route in suckling mice or intrathoracic inoculation of mosquitoes. Newborn animals can develop encephalitis symptoms, but with some dengue strains, mice may exhibit no signs of illness. Virus antigen is detected in mouse brain or mosquito head squashes by staining with anti-dengue antibodies.

References

  • Centers for Disease Control and Prevention
  • Chatterji S, Allen JC Jr, Chow A, Leo YS, Ooi EE. Evaluation of the NS1 rapid test and the WHO dengue classification schemes for use as bedside diagnosis of acute dengue fever in adults. Am J Trop Med Hyg. 2011 Feb. 84(2):224-8.

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